Compositions and methods for therapeutic use

ABSTRACT

A method and pharmaceutical composition for the treatment of cancer using a gene delivery system, such as a viral vector delivery system, comprising a therapeutic gene such as p53 or a retinoblastoma tumor suppressor gene wherein the gene delivery system is formulated in a buffer comprising a delivery-enhancing agent such as ethanol or a detergent.

This application is a continuation-in-part of U.S. Ser. No. 08/584,077,filed Jan. 8, 1996.

BACKGROUND OF THE INVENTION

The present invention is directed to compositions and methods oftreating cancer by gene therapy using a therapeutic gene, such as atumor suppressor gene delivered by a gene delivery system, such as arecombinant viral vector delivery system, formulated in a buffercomprising a delivery-enhancing agent. In particular, this inventionrelates to the delivery of a tumor suppressor gene (e.g., p53 orretinoblastoma (RB)) to cancerous epithelial tissues and organs, such asthe bladder, using a recombinant adenoviral vector delivery systemformulated in a buffer comprising a delivery-enhancing agent.

Carcinoma of the bladder represents a significant source of morbidityand mortality. Bladder cancer ranks 10th in males and 12th in females incancer related mortality (Cancer Facts and Figures, Amer. Can. Soc. 5:11(1995)). Therapies available for the treatment of bladder cancer includeadjuvant chemotherapy or immunotherapy, transurethral resection ofsuperficial disease, radical cystectomy or radiotherapy which is oftencombined with systemic chemotherapy. Despite these therapeutic options,overall survival has not changed appreciably. (Ibid) Thus, newtherapeutic modalities must be developed for the treatment of bladdercancer.

Gene therapy strategies have been developed as an alternativetherapeutic approach (See for example, Brewster et al. Eur Urol25:177-182 (1994); Takahashi et al., Proc Natl Acad Sci USA 88:5257-5261 (1991); Rosenberg, S A, J. Clin Oncol. 10:180-199 (1992)).

Distinct approaches have been developed to treat neoplasms based on genetransfer methods. Methods have been developed to correct specificlesions at defined genetic loci which give rise to neoplastictransformation and progression (Spandidos et al., Anticancer Res. 10:1543-1554 (1990); Banerjee et al. Cancer Res. 52:6297-6304 (1992)).Overexpression of dominant oncogenes may be addressed using techniquesto inhibit the transforming gene or gene product. Loss of tumorsuppressor gene function may be approached using methods to reconstitutewild-type tumor suppressor gene function (Goodrich et al., Cancer Res.52:1968-1973 (1992)). Besides these methods to achieve mutationcompensation, genetic techniques have been developed to specifically andselectively eradicate tumor cells. These approaches of molecularchemotherapy rely on specific expression of toxin genes in neoplasticcells (Abe et al., Proc Soc Exp Biol Med. 203:354-359 (1993)). Finally,gene transfer methods have been used to achieve antitumor immunization.These methods of genetic immunopotentiation use techniques of geneticimmunoregulation to enhance immune recognition of tumors. Consequently,a variety of distinct approaches have been developed to accomplish genetherapy of cancer.

A high incidence of mutations has been observed in tumor suppressorgenes, such as p53 and RB, in the case of carcinoma of the bladder(Fujimoto et al. Cancer Res. 52:1393-1398 (1992); Cairns et al. Oncogene6:2305-2309 (1991)). For such genetic lesions of tumor suppressor genes,reversion of the neoplastic phenotype can be demonstrated withreplacement of the corresponding wild-type tumor suppressor gene(Spandidos, Id.; Banerjee, Id.).

In vitro studies using cell lines derived from human bladder tissueshave demonstrated efficient transgene expression following infectionwith recombinant adenovirus (Bass et al. Cancer Gene Therapy 2:2:97-104(1995)). Experiments in vivo have also shown adenovirus transgeneexpression in the urinary bladder of rodents after intravesicaladministration (Ibid; Morris et al. J. Urology. 152:506-50 (1994)). Invitro experiments with wild-type adenovirus demonstrate that virusattachment and internalization is not influenced by benzyl alcohol, butdo demonstrate an enhanced uncoating of the virion (Blixt et al. Arch.Virol. 129:265-277 (1993)). In vivo efforts with agents (e.g. acetone,DMSO, prolamine sulfate) can break down the protective “mucin” layerthat protects the bladder epithelium from bacteria, viruses and otherpathogens (Monson et al. J. Urol. 145:842-845 (1992); Parsons et al. J.Urol. 143:139-142 (1990)). None of the methods tried to date achieveenhanced delivery of a therapeutic tumor suppressor gene to the bladderfor the treatment of bladder cancer. In order to accomplish gene therapyfor treatment of bladder cancer, gene therapy methods must be developedto accomplish direct, optimal, in vivo tumor suppressor gene delivery tothe bladder epithelium.

These needs and others are addressed by the instant invention.

SUMMARY OF THE INVENTION

One aspect of the invention is a method of administering a therapeuticagent to a tissue having an epithelial membrane, comprisingadministering a therapeutically effective amount of the therapeuticagent formulated in a buffer comprising a detergent.

A further aspect of the invention is a pharmaceutical compositioncomprising a therapeutically effective amount of the therapeutic agentformulated in a buffer comprising a detergent.

A further aspect of the invention is a method of treating bladder cancercomprising administration of a therapeutically effective amount of atherapeutic gene contained within a gene delivery system that isformulated in a buffer comprising a delivery-enhancing agent.

A further aspect of the invention is a pharmaceutical formulation foradministration of a recombinant adenovirus, comprising about 10⁹-10¹¹particles (PN)/ml recombinant adenovirus, about 2-10 mM Big CHAP orabout 0.1-1.0 mM TRITON®-X-100 detergent, phosphate buffered saline(PBS), about 2-3% sucrose (w/v) and about 1-3 mM MgCl₂, about pH6.4-8.4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the influence of formulation on adenovirus mediated genetransfer and expression in the rat bladder epithelium after intravesicaladministration.

FIG. 2 depicts adenovirus transgene expression in bladder epithelialcells after intravesical administration.

FIG. 3 depicts dose dependent adenovirus transgene expression in the ratbladder after intravesical administration.

FIG. 4 depicts a reverse-transcriptase polymerase chain reaction(RT-PCR) analysis of recombinant adenovirus transgene expression in themouse bladder after intravesical administration.

FIG. 5 depicts a time course of recombinant adenovirus transgeneexpression in bladder, kidney, and liver tissue after intravesicaladministration of the virus.

FIG. 6 depicts recombinant adenovirus transgene DNA in bladder andkidney homogenates after intravesical administration FIG. 7 depictsimprovement of gene transfer to bladder epithelium using a Big CHAP(N,N, bis-(3-D-gluconamidopropyl)-cholamide (CALBIOCHEM® Biochemicals)formulation.

FIG. 8 depicts improvement of gene transfer to bladder epithelium usingdifferent concentrations of recombinant adenovirus in a 7 mM Big CHAPformulation.

FIG. 9 depicts enhancement of recombinant adenovirus transgeneexpression in bladder tissue by using an ethanol (ETOH) or Big CHAPformulation.

FIG. 10 depicts gene transfer to tumors using a 4 mM Big CHAPformulation.

FIG. 11 depicts transgene transfer to pig bladder epithelium.

FIG. 12 depicts the expression of p53 in tumor tissue.

FIG. 13 depicts gene transfer to the muscosa of rat ileum.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a gene delivery system” refers to any means of deliveryof a therapeutic gene to a particular epithelial tissue or organincluding, for example, recombinant vectors and non-vector systems.Examples of non-vector systems include but are not limited to anylipid-based, lipid encapsulated DNA or cationic lipid/DNA complexes.Examples of recombinant viral vectors include but are not limited toherpes virus, retrovirus, vaccinia virus, adenovirus, andadenoassociated virus. “Recombinant” refers to nucleic acids and proteinencoded by them wherein the nucleic acids are constructed by methods ofrecombinant DNA technology, also termed “genetic engineering”. Apreferred recombinant viral vector is the adenoviral vector deliverysystem which has a deletion of the protein IX gene (See InternationalPatent Application WO 95/11984, which is herein incorporated byreference in its entirety for all purposes). The recombinant vectordelivery system comprising a therapeutic gene, such as a tumorsuppressor gene, is formulated in a buffer comprising adelivery-enhancing agent. “A delivery-enhancing agent” refers to anyagent which enhances delivery of a therapeutic gene, such as a tumorsuppressor gene to a cancerous tissue or organ. Such enhanced deliverymay be achieved by various mechanisms. One such mechanism may involvethe disruption of the protective glycosaminoglycan layer on theepithelial surface of the bladder. Examples of such delivery-enhancingagents are detergents, alcohols, glycols, surfactants, bile salts,heparin antagonists, cyclooxygenase inhibitors, hypertonic saltsolutions, and acetates. Alcohols include for example the aliphaticalcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetylalcohol. Glycols include glycerine, propyleneglycol, polyethyleneglycoland other low molecular weight glycols such as glycerol andthioglycerol. Acetates such as acetic acid, gluconol acetate, and sodiumacetate are further examples of delivery-enhancing agents. Hypertonicsalt solutions like 1M NaCl are also examples of delivery-enhancingagents. Examples of surfactants are sodium dodecyl sulfate (SDS) andlysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene,lysophosphatidylcholine, polyethylenglycol 400, polysorbate 80,polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bilesalts such as taurocholate, sodium tauro-deoxycholate, deoxycholate,chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid andother astringents like silver nitrate may be used. Heparin-antagonistslike quaternary amines such as prolamine sulfate may also be used.Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, andnon-steroidal antiinflammatory drug (NSAIDS) like indomethacin,naproxen, diclofenac may be used.

Detergents include anionic, cationic, zwitterionic, and nonionicdetergents. Exemplary detergents include but are not limited totaurocholate, deoxycholate, taurodeoxycholate, cetylpyridium,benalkonium chloride, ZWITTERGENT®3-14 detergent, CHAPS(3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate,Aldrich), Big CHAP, Deoxy Big CHAP, TRITON®-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent, TWEEN® 20 detergent,and TWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

In an embodiment, the delivery-enhancing agent is included in the bufferin which the recombinant adenoviral vector delivery system isformulated. The delivery-enhancing agent may be administered prior tothe recombinant virus or concomitant with the virus. In someembodiments, the delivery-enhancing agent is provided with the virus bymixing a virus preparation with a delivery-enhancing agent formulationjust prior to administration to the patient. In other embodiments, thedelivery-enhancing agent and virus are provided in a single vial to thecaregiver for administration.

In the case of a pharmaceutical composition comprising a tumorsuppressor gene contained in a recombinant adenoviral vector deliverysystem formulated in a buffer which further comprises adelivery-enhancing agent, the pharmaceutical composition may beadministered over time in the range of about 5 minutes to 3 hours,preferably about 10 minutes to 120 minutes, and most preferably about 15minutes to 90 minutes. In another embodiment the delivery-enhancingagent may be administered prior to administration of the recombinantadenoviral vector delivery system containing the tumor suppressor gene.The prior administration of the delivery-enhancing agent may be in therange of about 30 seconds to 1 hour, preferably about 1 minute to 10minutes, and most preferably about 1 minute to 5 minutes prior toadministration of the adenoviral vector delivery system containing thetumor suppressor gene.

The concentration of the delivery-enhancing agent will depend on anumber of factors known to one of ordinary skill in the art such as theparticular delivery-enhancing agent being used, the buffer, pH, targettissue or organ and mode of administration. The concentration of thedelivery-enhancing agent will be in the range of 1% to 50% (v/v),preferably 10% to 40% (v/v) and most preferably 15% to 30% (v/v).Preferably, the detergent concentration in the final formulationadministered to the patient is about 0.5-2× the critical micellizationconcentration (CMC). A preferred concentration of Big CHAP is about 2-20mM, more preferable about 3.5-7 mM.

The buffer containing the delivery-enhancing agent may be anypharmaceutical buffer such as phosphate buffered saline or sodiumphosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin the pharmaceutical composition comprising the tumor suppressor genecontained in the adenoviral vector delivery system, may be in the rangeof 6.4 to 8.4, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

A preferred formulation for administration of a recombinant adenovirusis about 10⁹-10¹¹ PN/ml virus, about 2-10 mM Big CHAP or about 0.1-1.0mM TRITON®-X-100 detergent, in phosphate buffered saline (PBS), plusabout 2-3% sucrose (w/v) and about 1-3 mM MgCl₂, at about pH 6.4-8.4.

The term “enhanced” describes the increased delivery of the therapeuticgene, such as a tumor suppressor gene, to the cancerous tissue or organ.Increased delivery of a therapeutic gene, such as a tumor suppressorgene, can be measured by various means, for example by measuringexpression of the tumor suppressor gene compared to expression levelswhen the tumor suppressor gene is delivery in an adenoviral vectordelivery system in a buffer lacking the delivery-enhancing agent.Examples of therapeutic genes are tumor suppressor genes and the suicidegene thymidine kinase. Examples of tumor suppressor genes include butare not limited to p53, the retinoblastoma gene, either full length(p110^(RB)) or fragments thereof such as p94^(RB) or p56^(RB), and p16.Other therapeutic genes include but are not limited to CFTR, genesencoding cytokines (such as the interferons α, β, γ, δ, interleukins(e.g., IL-4, IL-10, IL-2), GM-CSF, and any other genes encoding proteinswhich have therapeutic potential in the treatment of non-cancerousdiseases of the bladder such as cystitis. In some embodiments of theinvention, the therapeutic gene encodes antisense RNA.

In some embodiments, the compositions of the invention comprise atherapeutically effective amount of a therapeutic gene, such as a tumorsuppressor gene contained in a recombinant viral vector delivery systemin a buffer comprising a delivery-enhancing agent. “Therapeuticallyeffective” as used herein refers to the prevention of, reduction of, orcuring of symptoms associated with a disease state.

Therapeutically effective amounts of the pharmaceutical compositioncomprising a therapeutic gene, such as p53 or the retinoblastoma tumorsuppressor gene, in a recombinant viral vector delivery systemformulated in a buffer comprising a delivery-enhancing agent will beadministered in accord with the teaching of this invention. For example,therapeutically effective amounts of the retinoblastoma tumor suppressorgene in the recombinant adenoviral vector delivery system formulated ina buffer containing a delivery-enhancing agent are in the range of about1×10¹¹ particles/ml to 1×10¹² particles/ml, more typically about 1×10⁸particles/ml to 5×10¹¹ particles/ml, most typically 1×10⁹ particles/mlto 1×10¹¹ particles/ml (PN/ml).

The compositions of this invention may additionally include astabilizer, enhancer or other pharmaceutically acceptable carriers orvehicles. A pharmaceutically acceptable carrier can contain aphysiologically acceptable compound that acts, for example, to stabilizethe recombinant adenoviral vector delivery system comprising the tumorsuppressor gene. A physiologically acceptable compound can include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Otherphysiologically acceptable compounds include wetting agents, emulsifyingagents, dispersing agents or preservatives, which are particularlyuseful for preventing the growth or action of microorganisms. Variouspreservatives are well known and include, for example, phenol andascorbic acid. One skilled in the art would know that the choice ofpharmaceutically acceptable carrier, depends on the route ofadministration and the particular physiochemical characteristics of therecombinant adenoviral vector delivery system and the particular tumorsuppressor gene contained therein. Examples of carriers, stabilizers oradjuvants can be found in Martin, Remington's Pharm. Sci., 15th Ed.(Mack Publ. Co., Easton, Pa. 1975), incorporated herein by reference.

The recombinant viral vector delivery system comprising a therapeuticgene formulated in a buffer comprising a delivery-enhancing agent may bedelivered to any cancerous tissue or organ using any delivery methodknown to the ordinarily skilled artisan for example, intratumoral orintravesical administration. Cancerous tissues and organs include anytissue or organ having an epithelial membrane such as thegastrointestinal tract, the bladder, respiratory tract, and the lung.Examples include but are not limited to carcinoma of the bladder andupper respiratory tract, vulva, cervix, vagina or bronchi; localmetastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleuralmetastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma ofthe nasopharynx, nose, larynx, oesophagus, stomach, colon and rectum,gallbladder, or skin; or melanoma.

The delivery-enhancing agents of the invention can also be used toformulate other pharmaceutical agents, such as proteins, nucleic acids,antisense RNA, small molecules, etc., for administration to any tissueor organ having an epithelial membrane.

The following examples are intended to illustrate, not limit the scopeof this invention.

EXPERIMENTAL EXAMPLES Example 1 Ethanol Improves Gene Transfer in theBladder

Initial experiments have shown that several factors including virusconcentration, time of administration, and volume of dosing caninfluence gene transfer to the bladder epithelium after intravesicaladministration to rats. Because increased penetration of dyes can beachieved by intravesical administration of different solvents,modification of the adenovirus formulation was also investigated as analternative strategy to increase adenovirus transgene expression in thebladder (Monson et al. Urology 145:842-845 (1991)). The instantexperiments focused on the use of ethanol to increase adenovirustransgene expression in the bladder.

Nine female buffalo rats (Harlan Sprague Dawley) were anesthetized withisoflurane and received a single intravesical administration of a humanrecombinant adenovirus encoding the lacZ gene (rAd-βgal). The humanrecombinant adenoviral vector comprising the lacZ gene (rAd-βgal) isdescribed in Wills et al. Human Gene Therapy 5:1079-1088 (1994). Beforeinstillation bladders were flushed with PBS and emptied. rAd-βgal wasthen diluted to achieve a final concentration of 1.7×10¹¹ PN/mL in 1)VPBS (2% (w/v) sucrose and 2 mM MgCl, in PBS), 2) 30% (v/v) ethanol, or3) 50% (v/v) DMSO, and instilled in a 250 μL volume (N=3 animals/group).The administered material was retained in the bladder for 45 minutes.The bladder were then flushed with PBS, and the animals were permittedto recover from the procedure. Two days after administration, rats weresacrificed, bladders were harvested, fixed, and whole organs werestained with an Xgal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside)solution to evaluate reporter gene transfer. Xgal-stained tissues werethen paraffin embedded, sectioned, and counter stained with hematoxylinand eosin. Hydrolysis of Xgal by β-galactosidase results in a blue colorthat localized to the superficial luminal bladder epithelium.

Transgene expression, consequent to delivery by the adenoviral vector,was detected in bladders from all animals treated with rAd-βgal but notin an untreated control. Transgene expression was similar to previouslypublished results using the PBS/sucrose formulation (Bass et al. CancerGene Therapy 2:2:97-104 (1995)). In sharp contrast, β-galactosidaseexpression in the luminal epithelial surface was greatly enhanced inanimals that received rAd-βgal diluted in 30% ethanol (FIG. 1). Bladderspecimens described in FIG. 1 were embedded, sectioned, and counterstained with hematoxylin and eosin. Histologic evaluation of the bladdertissue demonstrated increased β-galactosidase expression of thetransitional bladder epithelium when ethanol was added to the adenovirusformulation (FIG. 2). The interaction of ethanol with the protectiveglycosaminoglycan (GAG) layer on the epithelium surface provides amechanism for the observed increase in transgene expression. Disruptionof this layer may facilitate virus-cell interaction at the surface andpotentially enhance penetration into the submucosa.

Example 2 Dose-Dependent Transgene Expression in the Rat Bladder

In another experiment, 18 female Sprague-Dawley rats were anaesthetizedwith isoflurane and received a single 0.5 ml intravesical bolus ofrAd-βgal at concentrations of 2×10⁷, 2×10⁸, 2×10⁹, 2×10¹⁰, and 2×10¹¹,PN/mL in a 22.5% (v/v) ethanol formulation. After a 45 minuteincubation, the bladders were flushed with PBS, and animals werepermitted to recover from anesthesia. Two days later, animals weresacrificed, and bladders were harvested, fixed, and whole organs werestained with Xgal solution to evaluate adenovirus transgene expression.β-galactosidase expression in the luminal bladder epithelium correlatedwith the concentration of the administered recombinant adenovirus (FIG.3). No striking differences were observed among animals receiving 2×10¹⁰or 2×10¹¹ PN/mL, suggesting a saturation of transgene expression in thismodel: Analysis of the volume voided after instillation indicated only aminimal reduction in the infectious titer of the dosing material atthese high doses. Expression of β-galactosidase decreased at lowerconcentrations. No evidence of β-galactosidase expression was detectedin animals dosed at a concentration of 1×10⁷ PN/mL or in an untreatedcontrol animal.

Example 3 ACNRB Gene Transfer in the Mouse Bladder

A pilot study was conducted to specifically evaluate expression of theRB transgene using a RT-PCR assay. The recombinant adenovirus used inthis study was based on serotype 5 human adenovirus from which the viralearly region 1 encoding E1a, E1b, and pIX proteins have been deleted.This adenovirus is limited to propagation in 293 cells which produce theAd5 El gene products required for replication. Transfer plasmidsencoding either full length or truncated Rb were generated from pACN(Wills et al. Cancer Gene Therapy 2:191-197 (1995)) and were, in turn,used to construct the recombinant adenoviruses. Either a full-length RBcDNA (1-928 amino acids), subcloned as a 2.8 Kb Xba I-Bam HI fragmentfrom the plasmids pETRbc (Huang et al. Nature 350:160-162 (1991) or atruncated fragment (amino acids 381-928) subcloned as a 1.7 KB Xba 1-BamHI cDNA fragment, was placed downstream of the CMV promoter/enhancer andthe Ad 2 tripartite leader cDNA of the plasmid pACN. These plasmids weresubsequently linearized with Eco RI and cotransfected (CaPO₄,Stratagene) with either the isolated Cla I digested large fragment ofH5ilE4 (Hemstrom et al. J. Virol. 62:3258-3264 (1988)), to make Ad-RB56(ACN56) containing a partial E4 deletion, or with the large fragmentfrom a hybrid virus of dl327 (Ginsberg et al. Proc. Natl. Acad. Sci.U.S.A. 86:3823-3827 (1989)) and H5ilE4 to create Ad-Rb110 (ACNRB) whichcontains deletions in both the E3 and E4 regions of the vector.

Eight female ICR mice (Charles River Laboratories) were anesthetizedwith avertine and each received a single 80 μl intravesicaladministration of (ACNRB). ACNRB (4×10¹¹ PN/mL) was diluted and preparedin a PBS solution or a 30% (v/v) ethanol solution. After the virus wasretained in the bladder for 45 minutes, the animals were permitted torecover and void. Mice were sacrificed 2 days or 14 days after ACNRBadministration, and bladders, livers, and kidneys from each animal wereharvested, homogenized, and processed for analysis (N=2 animals/group).Transgene expression was determined using RT-PCR with a primer specificfor ACNRB. More specifically, primers were generated to identify ACNRBand amplify the region from the 3′ end of the CMV sequence and to the 5′end of the RB sequence. Following amplification (30 cycles) RT-PCRproducts were separated on a 10% polyacrylamide gel, stained withethidium bromide, and photographed. Increased ACNRB expression wasdetected after treatment with ACNRB in 30% (v/v) ethanol compared tovery low expression after treatment with ACNRB in VPBS. Positivecontrols for the assay included samples from ACNRB-infected 5637 humanbladder cancer cells (CONTROL). Bladder RNA samples from ACNRB-infectedanimals that were amplified with primers specific for beta-actinprovided an internal control for the quality of RNA. Untreated samplesand bladder samples without the reverse transcriptase (RT) providedcontrols for contaminating DNA. Two days after dosing, levels of ACNRBexpression in the bladder homogenates were detected from animals thatreceived ACNRB prepared in 30% ethanol (FIG. 4). No evidence ofexpression was detected in non-bladder tissue or in any samplescollected 14 days after dosing.

Example 4 Kinetics of Biodistribution and ACNRB Expression AfterIntravesical Administration to Mice

To investigate the time course of expression after intravesicaladministration, 40 female mice (Charles River Laboratories) wereanaesthetized with avertine and received a single 80 μL bolus of ACNRB(4×10¹⁰ PN/mL in 22% (v/v) ethanol). The instilled material was retainedin the bladder for approximately 45 minutes, and animals were permittedto recover from the procedure. Mice were sacrificed 1, 2, 3, 4, 5, 6, 7,and 14 days after administration (N=4/time) for analysis. Bladders,livers, and kidneys were harvested and snap frozen in liquid nitrogenfor subsequent analysis. For detection of ACNRB expression, tissuesamples were homogenized, and total RNA was extracted usingTRI-Reagent®. An aliquot of total RNA was amplified in an RT-PCR assayusing primers specific for ACNRB to distinguish transgene expressionfrom endogenous RB expression. For detection of ACNRB DNA, a DNAextraction kit (Stratagene) was used on tissue homogenates. PCR wasperformed with the primers specific for ACNRB, as described above forthe RT-PCR analysis.

ACNRB transgene expression in the bladder homogenates was detected onlyin samples collected on days 1-6, with expression relative to endogenousp53 decreasing with time (FIG. 5, upper panel). No expression wasdetected from samples collected 7 and 14 days after administration.Interestingly, some ACNRB expression was detected in the kidneys on days1, 2 and 3, but no expression was observed in the liver (FIG. 5, lowerpanels).

ACNRB DNA was detected in bladder tissue of all animals that receivedACNRB, including those harvested 14 days after administration (FIG. 6,(left panel)). DNA was also recovered from the kidney homogenates,consistent with the ACNRB expression detected in this tissue (FIG. 6,right panel). No evidence for ACNRB DNA was detected in liver samplesharvested during the study (data not shown). Samples from an untreatedanimal (U) and purified ACNRB DNA (PC) were used as negative and 25positive controls, respectively.

Because systemic administration of recombinant adenovirus resultsprimarily in transgene expression in the liver (Li et al. Human GeneTherapy 4:403-409 (1993)), the absence of ACNRB DNA and expression inliver samples (FIGS. 5 and 6) suggests negligible systemic exposure ofACNRB after intravesical administration. Retrograde flow via the uretersmay have contributed to the detection of ACNRB in the kidney.

The data presented above demonstrate transgene expression in the rodentbladder following intravesical administration of ACNRB. These studiesfurther indicate that adenovirus-mediated gene transfer to the bladderepithelium can be enhanced by the presence of a delivery-enhancingagent, such as ethanol, in the formulation. One mechanism for theincreased gene transfer may be the disruption of the protectiveglycosaminoglycan layer on the epithelial surface of the bladder. Asingle intravesical administration of ACNRB in a 20-30% (v/v) ethanolformulation results in transgene expression in the bladder that persistsfor approximately one week. Retrograde ureteral flow provides a likelyexplanation for the transient expression of ACNRB detected in thekidney. The absence of ACNRB expression and ACNRB DNA in the liverindicates limited systemic exposure after intravesical administration.

Example 5 Use of Detergent Formulations

To minimize side effects without losing gene transfer efficiency, otherexcipients were tested. Detergents are known to interact with cellmembranes and form large pores without further damaging the cells. Theefficiency of recombinant adenovirus formulated in less toxic detergentswas studied in rats and mice gene transfer models.

rAd-βgal was formulated in different detergents at their criticalmicellization concentration to evaluate efficiency of gene transfer tothe bladder epithelium. Female rats (about 200 g b/w, Harlan SpragueDawley) were anesthetized with isoflurane and received a singleintravesical administration of rAd-βgal (1×10¹¹ PN/ml) in differentdetergent formulations (see Table I). Before instillation, bladders wereflushed with PBS and then emptied. rAd-βgal was then instilled in avolume of 0.5 ml. The instilled solution was retained in the bladder for45 minutes. The bladders were then flushed with PBS, and the animalswere permitted to recover from the procedure. 48 hours afteradministration, the rats were sacrificed, the bladders harvested, andfixed in formalin. After fixation, the bladders were openedlongitudinally so that the urothelium was exposed to the chromogen(Xgal), that is converted to a blue color, if reporter gene(β-galactosidase) expression is present. The luminal epithelial surfaceof the whole bladder was photographed an blue staining scored: +(minimalstaining), ++ (moderate staining), +++ intense staining covering thewhole bladder epithelial surface. The results are shown in Table I. Someof the anionic detergents (taurodeoxycholate), zwitterionic detergents(CHAPS, ZWITTERGENT®, and non-ionic detergents (Big CHAP, TRITON® X-100)enhanced gene transfer dramatically. Cationic detergents and some of thenonionic detergents (PLURONIC® F68, TWEEN®), did not have similareffects. In general, improvements of gene transfer were accompanied bycystitis. Zwiterionic detergents facilitated bladder stone formation.

Possible manifestations of cystitis as observed with ethanol wereevaluated in mice using a 7 MM Big CHAP (2×CMC) or 0.05 mM TRITON®-X-100detergent (CMC) formulation. The formulations were administeredintravesically in a volume of 80 uL, and animals were observed over a7-day interval. After sacrifice, bladders were paraffin-embedded,sectioned, and stained with hematoxylin and eosin for pathologicevaluation. Only a slight macrophage infiltration into the bladdertissue was observed in mice treated with Big CHAP. Macrophagesinfiltrated more prominently (slight to mild) induced by TRITON®-X-100detergent. In sharp contrast, significant cystitis was detected inanimals treated with 22% ethanol.

Dose Gene Expression in Excipient Charge of Detergent (nM) BladderEpithelium Gross Pathology Stability Taurocholate anionic 6 + none NDDeoxycholate anionic 5 + Cystitis ND Taurodeoxycholate anionic 6 +++Cystitis + Cetylpyridinium cationic 0.9 + none − Benzalkonium Chloridecationic 0.5% <+ none − Zwittergent ® 3-14 zwitterionic 4 +++ stoneformation ND Chaps zwitterionic 7 +++ stone formation + Big Chap nonionic 3.5 +++ none + Deoxy Big Chap non ionic 1.5 +++ Cystitis ND TritonX-100 non ionic 0.05 +++ none + C12E8 non ionic 4 ++ none NDOctyl-β-D-Glucopyranoside non ionic 10 ++ none ND Pluronic F68 non ionic0.04 + none + Tween 20 non ionic 2 + none + Tween 80 non ionic 0.02 +none ND Tween 80 non ionic 2 + none +

Example 6 Gene Transfer of ACNRB

In addition to the experiments with the reporter gene, a different setof studies was conducted to specifically evaluate gene transfer ofACNRB. Female ICR mice were anesthetized with avertine and each mousereceived a single 80 μL intravesical administration of ACNRB. ACNRB(4×10¹⁰ PN/mL) was formulated in VPBS, 22% (v/v) ethanol, or 3 mM BigCHAP. After the virus was retained in the bladder for 45 minutes, theanimals were permitted to recover. Mice were sacrificed 48 hours afterACNRB administration, and bladders snap frozen in liquid nitrogen.Transgene expression was determined using RT-PCR. Tissues were rinsed inRNAse free water, homogenized, digested in Tri-Reagent (MolecularResearch Center), and total cellular RNA extracted. ACNRB was probedusing a 5′ primer located in the CMV region of ACNRB vector, and a 3′primer resided in the 5′ end of Rb genome. RT-PCR was performed in thePerkin Elmer 9600 GeneAmp PCR System. Cycling conditions were 10 min at65° C., 8 min at 50° C., 5 min at 95° C. 32 cycles of PCR wereperformed, each cycle consisting of 30 sec at 94° C., 30 sec at 58° C.,and 30 sec at 72° C. The 32nd cycle included a 10 min elongation step at72° C. to ensure full extension of incomplete DNA fragments. ACNRB-RNAbands were stained with ethidium bromide. The results, enhancedexpression using an ethanol or Big CHAP formulation, are shown in FIG.9.

Example 7 Big CHAP Enhances Transgene Expression With Minimal Cystitis

Because Big CHAP enhanced gene transfer with minimal cystitis, thisformulation was selected for further evaluation, including concentrationand dose-dependence in studies similar to those described above.Briefly, in anaesthetized female rats rAd-βgal (1×10 ¹¹ PN/ml) wasadministered into the bladder via an intravesical catheter. rAd-βgal wasformulated in different concentrations of Big CHAP. A volume of 0.5 mlwas injected and remained instilled in the bladder for 45 minutes. Theanimals were sacrificed 48 hours later, the bladder fixed in 4%formalin/glutaraldehyde, opened longitudinally, and the β-galactosidaseenzyme activity measured using Xgal substrate. The intensity of bluestaining correlates with the βgal-transgene expression. The figure showsthe epithelial surface of Xgal stained bladders. The results indicate aconcentration-dependent increase of gene transfer to the epithelium. The3.5-7 mM concentrations of Big CHAP significantly improved genetransfer. The formulation alone (FIG. 7, lower panel) did not induce ablue color from the Xgal substrate. A higher concentration (17.5) mM didnot notably improve gene transfer or expression, but induced cystitis insome of the animals tested.

Effects of higher recombinant adenovirus concentrations were alsotested. Briefly, in anaesthetized female rats different concentrationsof rAd-βgal, formulated in 7 mM Big CHAP were administered into thebladder via an intravesical catheter. The animals were sacrificed 48hours later, the bladder fixed in 4% formalin/glutaraldehyde, openedlongitudinally, and Xgal stained. FIG. 8 shows a concentration dependentincrease of gene transfer to the epithelium. A concentration of 1.3×10¹¹PN/ml induced maximal gene transfer. A higher concentration (6.5×10¹¹PN/ml) did not notably improve the blue staining. In lowerconcentrations of rAd-βgal, 1.3×10¹⁰ PN/ml, or 1.3×10⁹ PN/ml, transgeneexpression reduced dose dependently. When 3.5 mM and 7 mM formulationswere compared, β-galactosidase expression was similar, although theenhanced effect appeared more reproducible in animals treated with the 7mM Big CHAP formulation.

Example 8 Transgene Expression in Tumors with Big CHAP Formulation

Because initial investigations focused on animals with intact bladderepithelium, evaluated adenovirus mediated gene transfer in an animalmodel of transitional cell carcinoma was also studied. Tumors wereinduced in male Fisher rats by addition of 0.05% BBN in the drinkingwater for six months. rAd-βgal (1×10¹¹ PN/ml), formulated in 4 mM BigCHAP or VPBS was instilled into the bladder for 45 minutes by directinjection. β-gal expression was evaluated 48 hr after treatment.Consistent with earlier experiments using non-tumor bearing animals,gene transfer to tumor tissue was improved with the Big CHAP formulationcompared to the VPBS formulation (FIG. 10).

Gene transfer of rAd carrying the p53 gene (rAd-p53) (Wills et al. HumanGene Therapy 5:1079-1088 (1994)) was also tested in this animal model ofbladder cancer. Briefly, bladder tumors were induced in female Fisherrates (Charles River) by addition of 0.05% BBN(N-butyl-N—N(4-hydroxybutyl)nitrosamine) in the drinking water for threemonths. rAD-p53 (1×10¹¹ PN/ml) was formulated in 7 mM Big CHAP. Underisoflurane anesthesia a catheter (24G) was inserted into the bladder foradministration. rAD-p53 was instilled into the bladder for 45 minutes.The animals were then allowed to recover from anesthesia. Twenty-four hrlater, animals were sacrificed, and the bladder was fixed in formalin.After paraffin embedding and sectioning, p53 expression was assayed byimmunohistochemistry using p53ES-kit (Oncogene) using AEC (AEC-kit,Vector Labs) as a substrate. Tissues were counterstained withhematoxylin. FIG. 12 shows p53 gene expression in the surface area ofproliferative epithelium (left panel) and nuclear staining for p53expression at higher magnification (right panel). No staining wasdetected in tumor tissue from untreated animals.

Example 9 Big CHAP Enhances Transgene Expression in Pig Urothelium

To simulate volumes expected for clinical investigation, the 7 mM BigCHAP formulation was tested in a chronically catheterized adult pigmodel in collaboration with SPRI Drug Safety and Metabolism. rAd-βgal(1×10¹¹ PN/ml) was formulated in VPBS or 7 mM Big CHAP. A volume of 50ml was injected via the catheter into the bladder of the consciousanimals. The instilled material was retained for 2 hr. The animals weresacrificed 48 hr later, and a central section of the bladder washarvested and stained for β-galactosidase expression. An increase in theintensity of gene expression was observed in the 7 mM Big CHAP treatedpig compared to the VPBS treated pig (FIG. 11). Histologic evaluationdemonstrated transduction of several epithelial layers using Big CHAP(left panel), but only superficial transduction with the VPBS buffer(right panel).

Example 10 Gene Transfer into Intestinal Epithelium in Rats

A slightly modification of the method of Sandberg et al. (Human GeneTherapy 5:323-329 (1994)) was used to prepare rat ileal segments forgene transfer studies. Briefly, female Sprague-Dawley rats wereanesthetized with isoflurane. The abdominal cavity was opened and anileal segment rostral from the last Peyer's patch isolated. The segment(about 3 cm) was cautiously cleared from food residues and both sidesclosed with atraumatic vascular clamps. rAd-βgal (1×1011 PN/ml), 0.5 mlvolume, was directly injected into the segment with a 24 G needle andallowed to incubate for 45 minutes. rAd-βgal was formulated in 10 mMtaurodeoxycholatic acid (in distilled water, sterile filtered)(Treatment group 1) or VPBS (Treatment Group 2). A third treatment groupcomprised animals treated with 10 mM taurodeoxycholatic acid.Thereafter, clamps were removed and a loose silk suture anchored on bothends for recognition at time of necropsy. The abdominal incision wasclosed and animals allowed to recover in their cages. Animals weresacrificed 48 hr later. The infected segment and a control segment wereharvested in fixative for whole organ Xgal staining.

The results are shown in FIG. 13. The extent of Xgal blue stainingdemonstrated evidence of transgene expression in the ileal sections.Enhanced gene transfer was evident in the detergent formulation (medialpanel).

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above, without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

1. A method of administering a therapeutic agent to the urinary bladdercomprising intravesically administering to the urinary bladder atherapeutically effective amount of the therapeutic agent formulated ina buffer comprising a detergent.
 2. The method of claim 1, wherein thetherapeutic agent is a protein.
 3. The method of claim 1, wherein thetherapeutic agent is a nucleic acid.
 4. The method of claim 3, whereinthe nucleic acid is a tumor suppressor gene.
 5. The method of claim 1,wherein the detergent is Big CHAP.
 6. (canceled)
 7. The method of claim3, wherein the nucleic acid encodes a cytokine.
 8. The method of claim7, wherein the cytokine is an interferon α, β, γ, or δ. 9-10. (canceled)11. A pharmaceutical composition comprising a therapeutically effectiveamount of a therapeutic agent formulated in a buffer comprising adetergent.
 12. The pharmaceutical composition of claim 11, wherein thetherapeutic agent is a protein.
 13. The pharmaceutical composition ofclaim 11, wherein the therapeutic agent is a nucleic acid.
 14. Thepharmaceutical composition of claim 13, wherein the nucleic acid is atumor suppressor gene. 15-16. (canceled)
 17. The pharmaceuticalcomposition of claim 11, wherein the detergent is Big CHAP. 18.(canceled)
 19. (canceled)
 20. A method of treating bladder cancercomprising intravesically administering to the urinary bladder of asubject having the bladder cancer a therapeutically effective amount ofa therapeutic gene contained within a gene delivery system that isformulated in a buffer comprising a delivery-enhancing agent.
 21. Themethod of claim 20 wherein the gene delivery system is a viral vectordelivery system.
 22. The method of claim 20 wherein the gene deliverysystem is a recombinant adenoviral vector delivery system.
 23. Themethod of claim 20 wherein the therapeutic gene is a tumor suppressorgene. 24-27. (canceled)
 28. The method of claim 20 wherein thedelivery-enhancing agent is a detergent.
 29. The method of claim 28,wherein the detergent is Big CHAP. 31-39. (canceled)
 40. The method ofclaim 20, wherein the gene encodes a cytokine.
 41. The method of claim20, wherein the gene encodes an interferon α, β, γ, or δ.